CN107966502B - Method for determining polysaccharide hydrolysate-galacturonic acid in salvia miltiorrhiza bunge - Google Patents
Method for determining polysaccharide hydrolysate-galacturonic acid in salvia miltiorrhiza bunge Download PDFInfo
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Abstract
The invention discloses a method for determining a salvia polysaccharide hydrolysate-galacturonic acid by adopting an HPLC method, which supplements the existing method for detecting compound salvia tablets and comprises the following steps: (1) collecting a compound salvia tablet sample, (2) defining chromatographic conditions, (3) determining a correction factor, (4) preparing a sample solution, (5) inspecting the linear relation of the stability of the detected solution, the galacturonic acid sample amount and a peak area ratio, and carrying out accuracy, precision and reproducibility tests, and (6) determining the content of galacturonic acid in the sample. The invention has high accuracy, good precision and good reproducibility, and provides a detection basis for detecting that the salvia miltiorrhiza in the compound salvia tablet is extracted only by ethanol with proper concentration without extracting according to a standard process.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for determining a polysaccharide hydrolysate, namely galacturonic acid, of salvia miltiorrhiza, and belongs to a supplementary inspection method of compound salvia miltiorrhiza tablets.
Background
The prescription of the compound salvia tablet comprises three medicinal materials of salvia miltiorrhiza, panax notoginseng and borneol, wherein the salvia miltiorrhiza is extracted by 95 percent ethanol and 50 percent ethanol in sequence and is concentrated into extract after being decocted by water, and the panax notoginseng fine powder and the borneol are directly used as medicine. It is known that most of the salvia extracts used for feeding compound salvia tablets in the current market are the salvia which is extracted by ethanol with proper concentration, and the tanshinone IIAThe salvia extract with the content of 0.3 percent and the content of salvianolic acid B of 7.0 percent is added, thus meeting the requirements of salvia [ identification ] and [ content determination ] in the current pharmacopoeia standard of compound salvia tablets. However, compared with the standard process of the salvia in the compound salvia tablet, the salvia extract lacks the water decoction step and has low polysaccharide content. Therefore, further supplementary examination is required for the salvia miltiorrhiza [ identification ] and [ content determination ] requirements in the current pharmacopoeia standards.
Disclosure of Invention
Based on the method, the invention provides a method for measuring the polysaccharide hydrolysate-galacturonic acid of the salvia miltiorrhiza, which adopts an HPLC method to measure the content of the polysaccharide hydrolysate-galacturonic acid of the salvia miltiorrhiza. The invention has high accuracy, good precision and good reproducibility, and provides a detection basis for detecting that the salvia miltiorrhiza in the compound salvia tablet is extracted only by ethanol with proper concentration without extracting according to a standard process.
In order to achieve the technical purpose, the specific contents are as follows:
1. instruments and reagents:
AgiLent 1100 liquid chromatograph, variable wavelength detector; a DAD detector; waters UPLC liquid chromatograph, variable wavelength detector;
galacturonic acid control: the method is provided by China institute for testing and testing biological products, and comprises the following steps: 111646-;
glucosamine hydrochloride control: the method is provided by China institute for testing and testing biological products, and comprises the following steps: 140649-;
acetonitrile is chromatographically pure, water is high-purity water, and other reagents are analytically pure.
2. The measurement method is as follows:
(1) collecting a compound salvia tablet sample;
(2) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution (pH value adjusted to 6.9 by 40% sodium hydroxide solution) (18: 82) as mobile phase; the detection wavelength was 250 nm. The number of theoretical plates is not less than 3000 calculated according to galacturonic acid peak;
(3) and (3) determination of a correction factor: taking a proper amount of glucosamine hydrochloride, precisely weighing, and adding water to prepare a solution containing 1mg per 1mL as an internal standard solution; taking about 10mg of galacturonic acid reference substance, placing the galacturonic acid reference substance into a 20mL measuring flask, adding a proper amount of water to dissolve and dilute the galacturonic acid reference substance to a scale, precisely sucking 2mL, placing the galacturonic acid reference substance into the 10mL measuring flask, precisely adding 1mL of internal standard solution, adding water to dilute the galacturonic acid reference substance to the scale, shaking up, sucking 400 mu L, adding 400 mu L of 1-phenyl-3-methyl-5-pyrazolone methanol solution of 0.5mol/L and sodium hydroxide solution of 0.3mol/L, uniformly mixing, and carrying out water bath reaction at 70 ℃ for 100 minutes. Adding 500 mu L of 0.3mol/L hydrochloric acid solution, mixing uniformly, washing with trichloromethane for 3 times, 2mL each time, discarding trichloromethane solution, adding 1 drop of phenolphthalein indicator solution into a water layer, adjusting the pH value to be neutral by using 0.3mol/L sodium hydroxide solution, centrifuging, precisely absorbing 0.7mL, precisely adding 0.7mL of phosphate buffer solution (pH 12, taking 50mL of 0.05mol/L disodium hydrogen phosphate solution, adding 26.9mL of 0.1mol/L sodium hydroxide solution, diluting with water to 100 mL), mixing uniformly, absorbing 20 mu L, injecting into a liquid chromatograph, measuring, and calculating a correction factor;
(4) preparation of a test solution: taking 20 tablets, removing the coating, precisely weighing, grinding, taking the amount corresponding to 5 tablets, precisely weighing, precisely adding 20mL of water, carrying out ultrasonic treatment (power 600W and frequency 46 kHz) for 1 hour, centrifuging (the rotating speed is 10000 rpm) for 5 minutes, precisely absorbing 5mL of supernatant, adding 5mL of absolute ethyl alcohol, shaking uniformly, standing at 4 ℃ for 1 hour, centrifuging, discarding the supernatant, adding 50% ethanol for washing 3 times, 2mL each time, centrifuging (the rotating speed is 10000 rpm) for 10 minutes, discarding the supernatant, adding 1mL of internal standard solution into the precipitate, adding water for dissolving, transferring to a 5mL measuring flask, diluting to the scale with water, and shaking uniformly. Sucking 1mL of the solution, placing the solution in a headspace bottle, adding 0.5mL of 3.0mol/L hydrochloric acid solution, sealing, uniformly mixing, hydrolyzing at 110 ℃ for 2 hours, cooling, and adjusting the pH value to be neutral by using 3.0mol/L sodium hydroxide solution;
(5) observing the stability of the solution to be tested and the linear relation between the galacturonic acid sample amount and the peak area ratio, and carrying out accuracy, precision and reproducibility tests;
(6) and (3) determination: sucking 400 mu L of test solution, adding 400 mu L of each of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone methanol solution and 0.3mol/L sodium hydroxide solution, mixing uniformly, and reacting in 70 ℃ water bath for 100 minutes. Adding 500 mu L of 0.3mol/L hydrochloric acid solution, mixing uniformly, washing with trichloromethane for 3 times, 2mL each time, discarding the trichloromethane solution, adding 1 drop of phenolphthalein indicator solution into the water layer, adjusting the pH value to be neutral by using 0.3mol/L sodium hydroxide solution, centrifuging, precisely absorbing 0.7mL, precisely adding 0.7mL of phosphate buffer solution (pH 12, 50mL of 0.05mol/L disodium hydrogen phosphate solution is taken, 26.9mL of 0.1mol/L sodium hydroxide solution is added, diluting to 100mL by using water), mixing uniformly, absorbing 20 mu L, injecting into a liquid chromatograph, and measuring to obtain the result.
The invention also comprises a durability test of the compound salvia tablet, which adopts chromatographic columns and chromatographs of different brands to investigate.
The invention has the beneficial effects that:
the invention adopts a method for determining a salvia polysaccharide hydrolysate-galacturonic acid by an HPLC method to evaluate whether production enterprises produce according to a production process, and the determination result shows that the galacturonic acid content in the compound salvia tablet sold in the market is abnormally low, and the reason is that salvia is not produced according to the production process, namely salvia is not decocted by water. The invention has high accuracy, good precision and good reproducibility, further supplements and perfects the inspection of the compound salvia tablet on the basis of Chinese pharmacopoeia, provides inspection basis for the detection that the salvia in the compound salvia tablet is not extracted according to the standard process and is only extracted by ethanol with proper concentration, and is beneficial to standardizing the production process of the compound salvia tablet.
Drawings
FIG. 1 shows a graph of the frequency of galacturonic acid in 308 samples from 101 manufacturing plants;
FIG. 2 is a graph showing the fragmentation data for galacturonic acid in 308 samples from 101 manufacturing facilities.
Detailed Description
In order to describe the present invention in more detail, the present invention will be further described with reference to the following examples.
Examples, the contents are as follows:
1. instruments and reagents:
AgiLent 1100 liquid chromatograph, variable wavelength detector; a DAD detector; waters UPLC liquid chromatograph, variable wavelength detector;
galacturonic acid control: the method is provided by China institute for testing and testing biological products, and comprises the following steps: 111646-;
glucosamine hydrochloride control: the method is provided by China institute for testing and testing biological products, and comprises the following steps: 140649-;
acetonitrile is chromatographically pure, water is high-purity water, and other reagents are analytically pure.
2. The detection method comprises the following steps:
(1) 308 samples of 101 manufacturing facilities were collected.
(2) Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution (pH value adjusted to 6.9 by 40% sodium hydroxide solution) (18: 82) as mobile phase; the detection wavelength was 250 nm. The number of theoretical plates should not be less than 3000 calculated as galacturonic acid peak.
(3) And (3) determination of a correction factor: taking a proper amount of glucosamine hydrochloride, precisely weighing, and adding water to prepare a solution containing 1mg per 1mL as an internal standard solution; taking about 10mg of galacturonic acid reference substance, placing the galacturonic acid reference substance into a 20mL measuring flask, adding a proper amount of water to dissolve and dilute the galacturonic acid reference substance to a scale, precisely sucking 2mL, placing the galacturonic acid reference substance into the 10mL measuring flask, precisely adding 1mL of internal standard solution, adding water to dilute the galacturonic acid reference substance to the scale, shaking up, sucking 400 mu L, adding 400 mu L of 1-phenyl-3-methyl-5-pyrazolone methanol solution of 0.5mol/L and sodium hydroxide solution of 0.3mol/L, uniformly mixing, and carrying out water bath reaction at 70 ℃ for 100 minutes. Adding 500 mu L of 0.3mol/L hydrochloric acid solution, mixing uniformly, washing with trichloromethane for 3 times, 2mL each time, discarding the trichloromethane solution, adding 1 drop of phenolphthalein indicator solution into the water layer, adjusting the pH value to be neutral by using 0.3mol/L sodium hydroxide solution, centrifuging, precisely absorbing 0.7mL, precisely adding 0.7mL of phosphate buffer solution (pH 12, 50mL of 0.05mol/L disodium hydrogen phosphate solution is taken, 26.9mL of 0.1mol/L sodium hydroxide solution is added, diluting to 100mL by using water), mixing uniformly, absorbing 20 mu L, injecting into a liquid chromatograph, measuring, and calculating a correction factor.
(4) Preparation of a test solution: taking 20 tablets, removing the coating, precisely weighing, grinding, taking the amount corresponding to 5 tablets, precisely weighing, precisely adding 20mL of water, carrying out ultrasonic treatment (power 600W and frequency 46 kHz) for 1 hour, centrifuging (the rotating speed is 10000 rpm) for 5 minutes, precisely absorbing 5mL of supernatant, adding 5mL of absolute ethyl alcohol, shaking uniformly, standing at 4 ℃ for 1 hour, centrifuging, discarding the supernatant, adding 50% ethanol for washing 3 times, 2mL each time, centrifuging (the rotating speed is 10000 rpm) for 10 minutes, discarding the supernatant, adding 1mL of internal standard solution into the precipitate, adding water for dissolving, transferring to a 5mL measuring flask, diluting to the scale with water, and shaking uniformly. Sucking 1mL of the solution, placing the solution in a headspace bottle, adding 0.5mL of 3.0mol/L hydrochloric acid solution, sealing, mixing uniformly, hydrolyzing at 110 ℃ for 2 hours, cooling, and adjusting the pH value to be neutral by using 3.0mol/L sodium hydroxide solution.
(5) Stability study of the tested solutions: the sample solutions of the same lot (lot: L4A 037) were measured at regular intervals for 6 times, and as a result, the RSD of the area of the galacturonic acid peak measured 6 times was 1.81% (n = 6), and the RSD of the area of the glucosamine hydrochloride peak was 1.43% (n = 6), and the test showed that the test substance was stable within 18 hours.
(6) And (3) accuracy test: accurately weighing 16.48mg of galacturonic acid reference substance, placing the galacturonic acid reference substance in a 10mL measuring flask, adding water to dissolve and dilute the galacturonic acid reference substance to a scale, accurately sucking 1mL, 2mL and 3mL respectively, placing the galacturonic acid reference substance in a 5mL measuring flask, adding water to dilute the galacturonic acid reference substance to the scale, and obtaining reference substance solutions for sample application (i), ii and iii);
precisely weighing 0.75g 3 part, 1.5g 3 part and 2.25g 3 part of samples with known content (batch number: L4A037, content: 0.6545 mg/tablet, average tablet weight 0.3091 g), precisely adding 20mL of water, carrying out ultrasonic treatment (power 600W, frequency 46 kHz) for 1 hour, centrifuging (the rotating speed is 10000 r.m.) for 5 minutes, precisely absorbing 5mL of supernatant, adding 5mL of absolute ethyl alcohol, shaking up, standing at 4 ℃ for 1 hour, centrifuging, discarding the supernatant, adding 50% ethyl alcohol to wash the precipitate for 3 times, 2mL each time, centrifuging (the rotating speed is 10000 r.m.) for 10 minutes, discarding the supernatant, adding 1mL of internal standard solution to the precipitate, adding water to dissolve, transferring to a 5mL measuring flask, diluting to the scale with water, and shaking up. Sucking 1mL, placing in a headspace bottle, adding 0.5mL of 3.0mol/L hydrochloric acid solution, sealing, mixing, hydrolyzing at 110 ℃ for 2 hours, cooling, adding 0.5mL of sample reference solutions respectively, adjusting the pH value to be neutral by using 3.0mol/L sodium hydroxide solution, sucking 400 mu L, performing operation according to the method for measuring a correction factor from the point that 0.5mol/L PMP methanol solution is added, sucking 20 mu L, injecting into a liquid chromatograph, measuring, and calculating the recovery rate, wherein the average recovery rate is 99.47% and the RSD =2.58% (n = 9).
(7) And (3) precision test: the same batch of sample solution (batch number: L4A 037) was tested for 6 times continuously according to the chromatographic conditions described above, resulting in a galacturonic acid peak area RSD of 1.1% (n = 6), which indicated better precision.
(8) And (3) repeatability test: for the same lot (lot: L4a 037) sample, 6 copies were measured in parallel, and the average of the 6 copies was 0.655 mg/piece, RSD =3.0% (n = 6). The result shows that the method has better repeatability.
(9) And (3) linear relation investigation:
accurately weighing 20.22mg of galacturonic acid reference substance, placing in a 20mL measuring flask, adding water to dissolve and dilute to scale, shaking up, and making into reference substance stock solution. Precisely sucking the reference stock solutions 2mL, 1mL, 3mL, 5mL and 8mL respectively, placing in measuring bottles of 20mL, 10mL and 10mL respectively, adding water to dilute to the scale, shaking up to obtain reference solutions A, 7, 8, 9 and 10, precisely sucking the reference solutions A, 2mL, 1mL, 3mL, 5mL and 7mL respectively, placing in measuring bottles of 20mL, 10mL and 10mL respectively, adding water to dilute to the scale, shaking up to obtain reference solutions B, 3, 4, 5 and 6, precisely sucking the reference solutions B, 1mL and 3mL respectively, placing in measuring bottles of 10mL respectively, adding water to dilute to the scale, shaking up to obtain reference solutions 1 and 2. Precisely sucking 1mL of the reference substance solution, 2mL of the reference substance solution, 3mL of the reference substance solution, 4 mL of the reference substance solution, 5mL of the reference substance solution, 6 mL of the reference substance solution, 7mL of the reference substance solution, 8mL of the reference substance solution, 9mL of the reference substance solution, 10mL of the reference substance solution, precisely adding 1mL of the internal standard solution, adding water to dilute the internal standard solution to a scale, shaking up the internal standard solution, sucking 400 mu L of the internal standard solution, adding 0.5mol/L of 1-phenyl-3-methyl-5-pyrazolone methanol solution and 0.3mol/L of sodium hydroxide solution to 400 mu. Adding 500 mu L of 0.3mol/L hydrochloric acid solution, mixing uniformly, washing with chloroform for 3 times, 2mL each time, discarding chloroform solution, adding 1 drop of phenolphthalein indicator solution into water layer, adjusting pH value to be neutral with 0.3mol/L sodium hydroxide solution, centrifuging, precisely absorbing 0.7mL, precisely adding 0.7mL of phosphate buffer solution (pH 12, 50mL of 0.05mol/L disodium hydrogen phosphate solution is taken, 26.9mL of 0.1mol/L sodium hydroxide solution is added, diluting with water to 100 mL), mixing uniformly, absorbing 20 mu L, injecting into a liquid chromatograph, and measuring. And (3) drawing a standard curve by taking the sample amount of the reference substance as an abscissa (X) and taking the ratio of the integrated value of the area of the galacturonic acid peak to the integrated value of the area of the glucosamine hydrochloride peak as an ordinate (Y), wherein the regression equation is as follows:
Y=0.5418X-0.03224 r=0.9998
the results show that: when the sample amount of the galacturonic acid is 0.0202-16.176 mug, the sample amount and the peak area ratio have good linear relation.
(10) And (3) determination: sucking 400 mu L of test solution, adding 400 mu L of each of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone methanol solution and 0.3mol/L sodium hydroxide solution, mixing uniformly, and reacting in 70 ℃ water bath for 100 minutes. Adding 500 mu L of 0.3mol/L hydrochloric acid solution, mixing uniformly, washing with trichloromethane for 3 times, 2mL each time, discarding the trichloromethane solution, adding 1 drop of phenolphthalein indicator solution into the water layer, adjusting the pH value to be neutral by using 0.3mol/L sodium hydroxide solution, centrifuging, precisely absorbing 0.7mL, precisely adding 0.7mL of phosphate buffer solution (pH 12, 50mL of 0.05mol/L disodium hydrogen phosphate solution is taken, 26.9mL of 0.1mol/L sodium hydroxide solution is added, diluting to 100mL by using water), mixing uniformly, absorbing 20 mu L, injecting into a liquid chromatograph, and measuring to obtain a result.
And (3) durability test: the results are shown in Table 2, which were obtained by examining column chromatography and chromatograph chromatography of different brands (examined sample is Yunan white drug group, Inc., batch number: ZHA 1418).
TABLE 2 comparison of three C18 columns (specification: 5 μm, 4.6 mm. times.250 mm)
The results show that all three kinds of chromatographic columns selected above meet the requirements of the usability of the method system, so the number of theoretical plates is formulated as under item (2).
(11) And analyzing and researching the content of the galacturonic acid in 308 batches of samples of 101 enterprises and enterprises by adopting a frequency distribution graph and a data range respectively. The frequency profile of galacturonic acid in 308 samples was derived from the SPSS software (see fig. 1) and was found to be negatively distributed, indicating that some abnormal low data was present. And counting the half-lactose aldehyde acid data in a segmentation mode (see figure 2), finding that more samples with lower galacturonic acid content have more batches, and 54 samples with the galacturonic acid content less than or equal to 0.10 mg/tablet relate to 29 enterprises and have the lowest content of 0.01 mg/tablet. It is presumed that the content of galacturonic acid is abnormally low because salvia miltiorrhiza is not decocted with water.
Claims (2)
1. The method for determining the polysaccharide hydrolysate-galacturonic acid in the compound salvia tablet is characterized in that the method is applied to the test of whether the salvia in the compound salvia tablet is produced according to the standard process, and the method comprises the following steps:
(1) collecting a compound salvia tablet sample;
(2) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution is taken as a mobile phase; the detection wavelength is 250 nm; the number of theoretical plates is not less than 3000 calculated according to galacturonic acid peak; the ratio of acetonitrile to 0.05mol/L potassium dihydrogen phosphate in the acetonitrile-0.05 mol/L potassium dihydrogen phosphate solution is 18: 82, and the pH value is adjusted to 6.9 by using 40% sodium hydroxide solution;
(3) and (3) determination of a correction factor: taking a proper amount of glucosamine hydrochloride, precisely weighing, and adding water to prepare a solution containing 1mg per 1mL as an internal standard solution; taking about 10mg of galacturonic acid reference substance, placing the galacturonic acid reference substance into a 20mL measuring flask, adding a proper amount of water to dissolve and dilute the galacturonic acid reference substance to a scale, precisely sucking 2mL, placing the galacturonic acid reference substance into the 10mL measuring flask, precisely adding 1mL of internal standard solution, adding water to dilute the galacturonic acid reference substance to the scale, shaking up, sucking 400 mu L, adding 400 mu L of 1-phenyl-3-methyl-5-pyrazolone methanol solution of 0.5mol/L and sodium hydroxide solution of 0.3mol/L, uniformly mixing, and carrying out water bath reaction at 70 ℃ for 100 minutes; adding 500 mu L of 0.3mol/L hydrochloric acid solution, mixing uniformly, washing with trichloromethane for 3 times, 2mL each time, discarding trichloromethane solution, adding 1 drop of phenolphthalein indicator solution into a water layer, adjusting the pH value to be neutral by using 0.3mol/L sodium hydroxide solution, centrifuging, precisely absorbing 0.7mL, precisely adding 0.7mL of phosphate buffer solution with the pH value of 12, mixing uniformly, absorbing 20 mu L, injecting into a liquid chromatograph, measuring, and calculating a correction factor;
(4) preparation of a test solution: taking 20 tablets, removing the coating, precisely weighing, grinding, taking the amount corresponding to 5 tablets, precisely weighing, precisely adding 20mL of water, and carrying out ultrasonic treatment for 1 hour under the ultrasonic condition: power 600W, frequency 46 kHz; centrifuging for 5 minutes, precisely absorbing 5mL of supernatant, adding 5mL of absolute ethyl alcohol, shaking up, standing at 4 ℃ for 1 hour, centrifuging, discarding the supernatant, adding 50% ethanol into the precipitate for washing 3 times, 2mL each time, centrifuging for 10 minutes, discarding the supernatant, adding 1mL of internal standard solution into the precipitate, adding water for dissolving, transferring into a 5mL measuring flask, diluting with water to a scale, and shaking up; sucking 1mL of the solution, placing the solution in a headspace bottle, adding 0.5mL of 3.0mol/L hydrochloric acid solution, sealing, uniformly mixing, hydrolyzing at 110 ℃ for 2 hours, cooling, and adjusting the pH value to be neutral by using 3.0mol/L sodium hydroxide solution;
(5) observing the stability of the solution to be tested and the linear relation between the galacturonic acid sample amount and the peak area ratio, and carrying out accuracy, precision and reproducibility tests;
(6) and (3) determination: sucking 400 mu L of a test sample solution, adding 400 mu L of each of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone methanol solution and 0.3mol/L sodium hydroxide solution, uniformly mixing, and carrying out water bath reaction at 70 ℃ for 100 minutes; adding 500 mu L of 0.3mol/L hydrochloric acid solution, mixing uniformly, washing with trichloromethane for 3 times, 2mL each time, discarding trichloromethane solution, adding 1 drop of phenolphthalein indicator solution into a water layer, adjusting the pH value to be neutral by using 0.3mol/L sodium hydroxide solution, centrifuging, precisely absorbing 0.7mL, precisely adding 0.7mL of phosphate buffer solution with the pH value of 12, mixing uniformly, absorbing 20 mu L, injecting into a liquid chromatograph, and measuring to obtain a result.
2. The method for determining the salvia polysaccharide hydrolysate-galacturonic acid in the compound salvia tablet as claimed in claim 1, further comprising the compound salvia tablet durability test: different brands of chromatographic columns and chromatographs are respectively adopted for investigation.
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